Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The subject matter of the present invention is a novel vector and the use
thereof for producing a heterologous protein or a gene of interest, that
can be used, for example, in the context of an immunization or gene
therapy programme and concerns in particular a self-replicating vector
lacking an antibiotic-resistance gene, comprising a sequence encoding the
ccdA protein functionally linked to a first promoter, the sequence of the
Cer locus and a heterologous sequence, functionally linked to a second
promoter.

Claims:

1. A self-replicating vector devoid of any antibiotic-resistance gene,
comprising: a sequence encoding the ccdA protein functionally linked to a
first promoter, the sequence of the Cer locus, and a heterologous
sequence functionally linked to a second promoter.

2. The vector as claimed in claim 1, in which the first promoter is the
mob constitutive promoter.

3. The vector as claimed in claim 1, in which the second promoter is an
inducible promoter.

4. The vector as claimed in claim 3, in which the second promoter is the
T7 promoter.

5. The vector as claimed in claim 1, in which the heterologous sequence
encodes a vaccine antigen.

6. The vector as claimed in claim 5, in which the heterologous sequence
encodes the rEPA protein.

7. The vector as claimed in claim 1, in which the heterologous sequence
encodes a sequence that can be used in the context of gene therapy.

8. The vector as claimed in claim 1, in which the heterologous sequence
encodes an enzyme.

10. The prokaryotic cell as claimed in claim 9 corresponding to an E.
coli cell.

11. A method for producing a heterologous protein, the method comprising:
(a) inoculating an appropriate culture medium with cells as defined in
claim 9, (b) fermenter culturing the cells thus transformed in the
absence of antibiotic, and (c) recovering the heterologous protein
produced during step (b) from the supernatant or from the cell pellet.

12. The method, as claimed in claim 11, for producing a heterogeneous
protein in which the heterogeneous protein is rEPA.

13. A method for producing a self-replicating vector as defined in claim
1, the method comprising: (a) inoculating an appropriate culture medium
with prokaryotic cells expressing the ccdB protein and containing a
vector as defined above, (b) fermenter culturing the cell thus
transformed in the absence of antibiotic, and (c) recovering the vector
produced during step (b).

14. A method for constructing a self-replicating expression vector as
defined in claim 1, the method comprising: (a) constructing a
self-replicating vector comprising an antibiotic-resistance gene flanked
respectively by a sequence 1 and a sequence 2, in which the sequences 1
and 2 are two overlapping sequences of the sequence encoding the ccdA
protein, which, after homologous recombination, reconstitutes a
functional sequence, (b) linearizing said vector by using a restriction
enzyme which cleaves the vector only between the sequences 1 and 2, (c)
transforming a prokaryotic cell expressing the ccdB protein, and (d)
recovering the prokaryotic cells comprising the self-replicating vector.

Description:

[0001] The present invention relates to a new vector and to the use
thereof for the production of a heterologous protein or of a gene of
interest that can be used, for example, in the context of an immunization
or gene therapy program.

[0002] The vectors comprise at least one selectable marker, the presence
of which is necessary during their construction process and also during
the cell culture phase which results in their amplification and
optionally in the production of a protein of interest.

[0003] The selectable markers conventionally used are
antibiotic-resistance genes.

[0004] The risks potentially associated with the use of these resistance
genes, such as dissemination in the environment or transfer of the gene
to a pathogenic strain, have led health authorities to limit the use of
the latter. The presence of a gene for resistance to an antibiotic is
today considered to be a major drawback for use in humans.

[0005] Several alternative solutions have therefore been proposed. Mention
may, for example, be made of selection systems based on the
complementation of an essential gene. These systems all have the drawback
of requiring the construction of a specific strain deficient in said
essential gene and the obligatory use of defined media which do not
contain the product of the essential gene.

[0006] The objective of the present invention is to provide a new vector
which can be used on an industrial scale, which has the double advantage
of producing a high expression yield, this being the case in the absence
of any use of antibiotics, and which can therefore be used for large
volumes (for example, 1000-10 000 liters).

[0007] This system is therefore particularly advantageous in the context
of an industrial production of components that can be used in humans.

[0008] The present invention therefore provides a self-replicating vector
devoid of any antibiotic-resistance gene, comprising: [0009] a sequence
encoding the ccdA protein functionally linked to a first promoter, [0010]
the sequence of the Cer locus, and [0011] a heterologous sequence
functionally linked to a second promoter.

[0012] According to one particular embodiment, the first promoter is the
mob constitutive promoter.

[0013] According to another embodiment, the second promoter is an
inducible promoter, in particular the second promoter is the T7 promoter.

[0014] According to one particular embodiment, the heterologous sequence
encodes a vaccine antigen.

[0015] According to another particular embodiment, the heterologous
sequence corresponds to a sequence that can be used in the context of a
gene therapy.

[0016] According to yet another embodiment, the heterologous sequence
encodes an enzyme.

[0017] According to another aspect, the present invention relates to a
prokaryotic cell expressing the ccdB protein, comprising a vector as
defined above.

[0018] According to one particular aspect, said prokaryotic cell is an E.
coli cell.

[0019] According to another aspect, the present invention relates to a
method for producing a heterologous protein, comprising the steps of:

[0026] (b) fermenter culturing the cell thus transformed in the absence of
antibiotic, and

[0027] (c) recovering the vector produced during step (b).

[0028] According to another aspect, the present invention relates to a
method for constructing a self-replicating vector as defined above,
comprising the steps of:

[0029] (a) constructing a self-replicating vector comprising a functional
antibiotic-resistance gene flanked respectively by a sequence 1 and a
sequence 2, in which the sequences 1 and 2 are two overlapping sequences
of the sequence encoding the ccdA protein, which, after homologous
recombination, reconstitutes a functional ccdA sequence,

[0030] (b) linearizing said vector by using a restriction enzyme which
cleaves the vector only between the sequences 1 and 2,

[0048] According to a first aspect, the present invention therefore
relates to a self-replicating vector comprising: [0049] a sequence
encoding the ccdA protein, functionally linked to a first promoter,
[0050] the sequence of the Cer locus, and [0051] a heterologous sequence
functionally linked to a second promoter.

[0052] The term "self-replicating vector" is intended to mean a nucleic
acid molecule capable of replicating autonomously in a host cell. The
vector according to the invention may be a plasmid, a phagemid or a
bacteriophage. A self-replicating vector therefore comprises one or more
sequences directing or controlling the expression of the product of the
nucleic acid sequences contained in said vector. Such a vector therefore
comprises in particular an origin of replication that is functional in
the host cell transformed with said vector. The vector according to the
invention is advantageously a plasmid, in particular a plasmid capable of
replicating in an E. coli cell.

[0053] Any origin of replication conventionally used in self-replicating
vectors may be used in the context of the present invention. The origin
of replication confers a more or less high specificity with regard to the
host cell and conditions the number of copies of said vector. The origin
that may be used may be a single-copy origin, a low-copy-number origin or
a high-copy-number origin. In the context of the use of the vector for
the expression of a protein of interest or for the production of vectors
that can be used for DNA immunization or gene therapy, the origin of
replication is advantageously a high-copy-number origin (conventionally
understood to mean several hundred copies) such as colE1.

[0054] The self-replicating vectors according to the invention are vectors
devoid of antibiotic-resistance genes.

[0055] In the context of the present invention, the expression "vector
devoid of antibiotic resistance gene" is intended to mean a vector which
does not contain any antibiotic-resistance gene or which comprises all or
part of a nonfunctional antibiotic-resistance gene. Advantageously, the
vector according to the invention does not comprise any
antibiotic-resistance gene.

[0056] The vector according to the invention comprises as single
selectable marker, a sequence encoding a ccdA protein. The ccdA protein
functions in association with a host cell comprising a functional gene
encoding the ccdB protein.

[0057] The gene encoding the poison (ccdB) is introduced into the
bacterial chromosome of the host cell. It encodes a stable protein of
approximately 100 amino acids, which binds to gyrase, inducing death of
the bacterium.

[0058] The gene encoding the antidote (ccdA), for its part, encodes an
unstable protein of approximately 90 amino acids, which neutralizes the
poison protein. This ccdA gene is introduced into the vector according to
the invention under the control of a constitutive promoter.

[0059] The expression of the poison gene is under the control of a
promoter which can be strongly repressed by the antidote or the
poison-antidote complex. Consequently, when the vector is present in the
host cell, the poison is not produced. Moreover, when the vector is lost,
the antidote is degraded by a protease and the induced poison production
causes death of the host cell. This ccdA/ccdB selection system is
described in detail by Szpirer CY and Milinkovitch MC in Biotechniques.
2005 May; 38(5):775-81.

[0060] Reference may also be made to document WO 99/58652 for a detailed
description of this selection system.

[0061] Said sequence encoding the ccdA protein is functionally linked to a
first promoter. Any constitutive promoter conventionally used in vectors
may be used in the context of the present invention. A noninducible
promoter will advantageously be used. It is not necessary to use a strong
promoter. Furthermore, the promoter does not necessarily have to be
repressed by ccdB or the ccdB/A complex. Advantageously, the mob
constitutive promoter is used.

[0062] The vector according to the invention also comprises the sequence
of the Cer locus. Said sequence can be inserted into the vector in any
orientation and at any position. Said Cer sequence is described by
Summers D. K. and Sherratt, D. J. in EMBO J. 7 (3), 851-858 (1988). Said
Cer sequence is reproduced in the sequence listing in SEQ ID No. 1.

[0063] The vector according to the invention also comprises a heterologous
sequence functionally linked to a second promoter.

[0064] In the context of the present invention, the "heterologous
sequence" is intended to mean a sequence encoding a therapeutic protein
or a protein that can be used for diagnostic purposes or encoding a
vaccine antigen, and also encoding any protein of commercial or
industrial interest or a sequence of a heterologous gene that is of
interest in DNA vaccination or gene therapy.

[0065] By way of examples of therapeutic proteins, mention may in
particular be made of: blood derivatives, hormones, in particular growth
hormone, lymphokines, proteins encoding an enzyme activity capable of
converting a prodrug to a drug, in particular in the context of a cancer
treatment protocol, as described, for example, in Table 3 of the general
review by Kratz et al. ChemMedChem. 2008 January; 3(1):20-53.

[0066] By way of examples of a vaccine antigen, mention may be made of
proteins that can be used in an immunization program and also genes that
can be used in the context of a

[0068] By way of example of sequences that can be used in the context of a
gene therapy, mention may be made of the sequences that can be used in
the context of the treatment of genetic diseases such as cystic fibrosis.

[0069] A heterologous sequence encoding an enzyme, for example an enzyme
of industrial interest such as benzonase, trypsin or alternatively a
molecule capable of intervening in a biocatalytic process can be inserted
into a self-replicating vector according to the invention.

[0070] More specifically, mention may be made, by way of nonlimiting
example of a vaccine antigen that can be produced with the vector
according to the invention, of: Pseudomonas aeruginosa exoprotein A
detoxified, for example, by deletion of the Glu 553 residue (rEPA), the
tbpB (transferin binding protein) antigen of N. meningitidis B (Legrain
et al. Protein Expr Purif. 1995 October; 6(5):570-8), the Helicobacter
pilori AIpA protein, influenza virus hemaglutinin HA, such as, for
example, the sequence SEQ ID No. 5 or the corresponding sequences derived
from other flu virus strains, the sequence encoding CFTR, such as, for
example, SEQ ID No. 6, advantageously without introns (Babenko, A. P. J.
Biol. Chem. 283 (14), 8778-8782 (2008)), the antigens originating from
the "sporozoite" form of Plasmodium falciparum, (such as the major
sporozoite surface protein (circumsporozoite protein), LSA3 or the Pfs 16
antigen), and also antigens originating from the "merozoite" form of
Plasmodium falciparum (such as the MSP1, MSP2, MSP3, EBA-175, Rhop-1,
Rhop-2, Rhop-3, RAP-1, RAP-2, RAP-3, Pf155/RESA or AMA-1 antigen). The
sequences encoding these proteins are known and available on various
databases. For example, the complete sequence of the LSA 3 gene is 12240
base pairs long and encodes a protein of 1558 amino acids. The nucleotide
and protein sequences are described in the EMBL data bank under accession
numbers AE001424 and uniprot 096275-PLAF7. As heterologous sequence, use
may be made of the sequence encoding the whole protein for fragments of
this protein, such as those described in WO 02/38176. Use is customarily
made of the whole protein (which may contain one or more point mutations
so as to take into account the variations which exist between the strains
of Plasmodium falciparum) or a fragment of this protein of which the
amino acid sequence has at least 80%, in particular at least 90%,
especially at least 95% to 99% identity relative to the whole sequence
described in uniprot 096275-PLAF7.

[0071] The system according to the invention is particularly suitable for
the production of proteins for which glycosylation is absent or not
essential for their effectiveness or for the induction of an immune
response; on the other hand, sequences encoding glycosylated proteins may
be used, for example, in DNA vaccination.

[0072] Any constitutive or inducible promoter conventionally used in
vectors can be used in the context of the present invention as second
promoter. By way of example, mention may be made of the T7, T5,
arabinose, lac, Trp promoters or any other promoter derived from
microorganisms capable of functioning in a prokaryotic host cell, in
particular Escherichia coli. In the case of vectors used for gene therapy
or DNA immunization, the expression of the gene of interest may be under
the control of a promoter which functions in eukaryotic cells, such as
the CMV promoter or the SV40 promoter or alternatively promoters having a
cell specificity.

[0073] The vector according to the invention may also contain
expression-regulating sequences such as, for example, a
transcription-regulating terminator sequence, a signal sequence which
allows the exportation of the expressed protein to the periplasmic
compartment of the host cell or the secretion of said protein into the
culture supernatant of the host cells, and also a multiple cloning site.
These sequences are well known to those skilled in the art.

[0074] The vector according to the invention may be constructed by any
conventional genetic engineering technique.

[0075] Although use of the ccdA/ccdB selection is possible from the first
steps of construction of the vector, it is possible to maintain an
antibiotic-mediated selection pressure up to the final phase of the
vector construction method. The elimination of the antibiotic-resistance
gene can subsequently be carried out by simple digestion with a
restriction enzyme at unique sites, flanking the antibiotic-resistance
gene, and then religation of the vector on itself.

[0076] The final host cell/vector pair is obtained after transformation of
the prokaryotic cell with the plasmid that has been religated on itself.
A means of verifying the elimination of said gene then consists in
subculturing the colonies obtained, after transformation, in parallel, on
dishes containing or not containing the antibiotic, or alternatively in
verifying the absence of said gene by PCR, restriction mapping or any
other appropriate method. In any event, this additional verification
proves to be tedious.

[0077] The inventors have demonstrated another system for constructing the
vector according to the invention, which has the advantage of resulting
in easy selection of the clones sought.

[0078] According to a second aspect, a subject of the present invention is
therefore a method for producing a self-replicating vector as defined
above, comprising the steps of:

[0079] (a) constructing a self-replicating vector comprising an
antibiotic-resistance gene flanked, respectively, by a sequence 1 and a
sequence 2, in which the sequences 1 and 2 are two overlapping sequences
of the ccdA sequence, which, after homologous recombination,
reconstitutes a functional ccdA sequence,

[0080] (b) linearizing said vector by using a restriction enzyme having a
restriction site only between the sequences 1 and 2,

[0082] (d) recovering the cells containing the vector according to the
invention.

[0083] The new method according to the invention uses a recombination
event which makes it possible, in a one and only step, to eliminate the
antibiotic-resistance gene and to assemble a ccdA gene in its functional
form. In summary, the ccdA gene is cloned in the form of 2 separate and
individually nonfunctional elements located on either side of the
antibiotic-resistance gene. The parts referred to as 5' and 3' of ccdA
are defined in such a way as to contain a common sequence of, for
example, 200 nucleotides in length. As long as the antibiotic-resistance
gene is present, the ccdA assembly is nonfunctional. After digestion with
a restriction enzyme having a site located only between the sequences 1
and 2, for example within the antibiotic-resistance gene, a linear DNA
fragment is obtained. Through transfection of prokaryotic cells, for
example of competent E. coli cells, this DNA molecule can recircularize
by homologous recombination of the overlapping ccdA fragments. This step
makes it possible, on the one hand, to eliminate the
antibiotic-resistance gene and, on the other hand, to select the clones
containing the vector according to the invention, insofar as the ccdA
gene can be functional only after elimination of the
antibiotic-resistance gene. A schematic representation of the method
according to the invention is given in FIG. 2.

[0084] For the implementation of the method above, reference may be made
to a reference book in terms of genetic engineering, such as Maniatis et
al. "Condensed Protocols from Molecular Cloning: A Laboratory Manual" by
Joseph Sambrook and David W. Russell, which describes all the operating
conditions to be used for the steps for construction of a vector,
linearization, transformation and recovery of the clones. The
self-replicating vector can subsequently be readily recovered from the
transformed cells by any conventional method well known to those skilled
in the art.

[0085] As regards in particular the homologous recombination, the size of
the overlapping sequence can vary to a large extent. Sequences which
overlap over only 5 nucleotides can be used. The only important element
in the selection of the sequences 1 and 2 is that the two sequences,
after homologous recombination, reconstitute a functional ccdA sequence,
for example the complete ccdA sequence. Furthermore, they should flank
the antibiotic-resistance gene so as to allow elimination of the latter.

[0086] Any self-replicating vector as defined above can be advantageously
produced by the new method according to the invention.

[0087] According to another aspect, the present invention therefore
relates to a prokaryotic cell containing a self-replicating vector as
defined above.

[0088] Any prokaryotic cell expressing a functional ccdB protein can be
used in the context of the present invention. A ccdB protein is
functional if it is capable of significantly inhibiting the action of
gyrase, i.e. if it is capable of inducing a lethal effect on the
prokaryotic cell. It is therefore possible to use the whole ccdB protein
or fragments thereof which are capable of bringing about cell death in
the absence of ccdA protein. An E. coli cell expressing the ccdB protein,
in particular an E. coli cell in which the sequence encoding the ccdB
protein has been inserted into the bacterial genome, will advantageously
be used. The insertion of the ccdB sequence into the bacterial genome can
be carried out by any method well known to those skilled in the art for
performing such an insertion.

[0089] According to another aspect, the present invention therefore
relates to a method for producing a heterologous protein, comprising the
steps of:

[0095] (b) fermenter culturing the cell thus transformed in the absence of
antibiotic, and

[0096] (c) recovering the vector produced during step (b).

[0097] Conventionally, to implement these methods, the prokaryotic cells,
advantageously E. coli cells, originate from a freeze-dried material or a
frozen material; they are inoculated into a culture medium volume
generally not exceeding 1 liter. After an overnight period of culture or
when the optical density of the medium is sufficient, this first culture
is transferred into a second culture medium which is identical to or
different than the first, but the volume of which may be at least 10, in
particular at least 20 and especially at least 30 times or 60 times
greater. This second culture, inoculated between 1% and 10%, is carried
out in a fermenter of 1 to 10 000 liters, in particular in a fermenter of
30 to 10 000 liters, especially in a fermenter of 30 to 500 liters, such
as, for example, 50 to 100 liters.

[0098] In the case of the production of products of commercial interest,
such as enzymes, fermenters of larger volumes may be used. Fermenters of
1000 to 100 000 liters, for example 60 000 liters, may be used for this
last culture step. During the culture period, which is for example from 6
h to 24 h, a temperature of the order of 25° C. to 42° C.,
in general from 30 to 37° C., a pH of 6.5 and 7.5, shaking between
200 and 1500 rpm, a pressure of 0 to 500 mb, a pO2 adjusted between
20% and 50% and an equivalent air flow rate of 1 to 2 volumes of air per
volume of medium and per minute, which can be enriched in oxygen
representing 0 to 50% of the total gas flow, are customarily used.

[0099] Any culture medium suitable for the growth of the prokaryotic cell
used may be employed. A large number of culture media are described in
the literature and are commercially available. By way of example, mention
may be made of the E. coli cell culture media used in the context of the
present invention.

[0100] At the end of the exponential cell growth phase, it is possible to
further amplify the biomass by transferring it into another fermenter of
greater capacity using the same procedure. The culture volumes can reach
or even exceed 10 000 liters. The fermenter culturing step is generally
carried out according to the "batch" mode. It is also possible to adopt
other fermenter culturing modes such as the "fed batch" mode. In such a
situation, a nutritive supplement comprising carbohydrates is added to
the medium during the exponential growth phase so as to sustain the
bacterial multiplication and to obtain a higher cell density at the end
of the exponential phase. The amount of carbohydrate added is evaluated
according to the cell density and the cell growth rate. The fed batch
culturing mode is particularly advantageous in the context of the present
invention.

[0101] In the case of the production of a protein of interest, at the end
of culturing, if the protein of interest is secreted into the
supernatant, said supernatant is removed and the protein is purified. If
the protein is not secreted into the supernatant, the cell pellet is
recovered and the protein of interest is extracted and then purified from
said cell pellet. Any method described in the literature as being
suitable for this purpose can be used in the context of the present
invention. Reference may, for example, be made to the book "Protein
purification" 2nd edition, Janson, J-C & Ryden, L, 1998.

[0102] According to one particular embodiment, the prokaryotic strain used
is a strain of E. coli expressing the ccdB protein.

[0103] The method is advantageously used for the production of the
proteins identified above, in particular for the production of rEPA.

[0104] In the case of the production of a vector according to the
invention, the vector is recovered from the cell pellet and purified. Any
known method conventionally used in the literature for this purpose can
be used in the context of the present invention.

[0105] The inventors have demonstrated that the new vector according to
the invention results in a significant improvement in the amount of
proteins of interest or of vector of interest produced. The vector
according to the invention results in an improved containment in the
prokaryotic cell expressing the ccdB protein.

[0106] The containment of the vector in a prokaryotic cell can be readily
evaluated, for example, by determination, at a time t of the culture, of
the proportion of cells no longer containing the vector. Conventionally,
this determination is carried out by PCR evaluation of the presence of
vector DNA and counting.

[0107] The expression "improved containment in the prokaryotic cell" is
intended to mean an at least 20% decrease in prokaryotic cells having
lost the vector, by comparison with a vector/prokaryotic cell system in
which the ccdA/Cer system is replaced with a conventional selection
system using an antibiotic-resistance gene, under the same culture
conditions after a culture period of at least 18 hours and using the same
method of determination. The decrease measured is advantageously at least
30%, in particular at least 50%, especially at least 70%.

[0108] The vector according to the invention results in an improved
production, after fermenter culturing, of proteins of interest or vectors
of interest.

[0109] The expression "improvement in the fermenter production" is
intended to mean an at least 10% increase in the amount of protein of
interest produced relative to the amount of total proteins, as
determined, after fermenter culturing for 18 h, by densitometric analysis
of an electrophoresis gel stained either with coomassie blue or with
silver nitrate, by comparison with a production carried out under the
same conditions but using an antibiotic-resistance gene as sole selection
system (without cer fragment). Such a test is described in the examples
which follow.

[0110] When one is interested in the production of the vector as such, for
example for DNA vaccination purposes, the expression "improvement in the
fermenter production" is intended to mean an at least 10% by weight
increase in the amount of vector DNA, as determined, after 18 h of
fermenter culturing of the host strain containing the vector of interest,
by quantitative DNA analysis by measurement at 260 nm on a purified
vector sample, by comparison with a production carried out under the same
conditions and quantitatively analyzed under the same conditions but
using an antibiotic-resistance gene as sole selection system (without cer
fragment).

EXAMPLE 1

Construction of a Vector According to the Invention Expressing the rEPA
Protein

[0111] First of all, the mob promoter and the ccdA gene (SEQ ID No. 7)
were amplified by PCR with the primers NotccdA+(SEQ ID No. 2) and
EcoRIccdA-(SEQ ID No. 3) from the plasmid pStaby1 (5932 by sold by the
company Delphi genetics) used as template under the following conditions:

[0114] The DNA fragments are purified by preparative 1% agarose gel
electrophoresis in 1× Tris-acetate-EDTA buffer. The bands
corresponding to the fragments of interest are cut out and the DNA is
recovered by electroelution and purified with the Qiaquik kit from the
company Qiagen.

[0115] The ccdA PCR fragment is subsequently digested with 45 U of NotI
and 30 U of EcoRI per 2250 ng of DNA. In parallel, 5 μg of plasmid
PM1816 (7195 bp) is digested with 30 U of NotI and 20 U of EcoRI enzyme.
The restriction enzymes from Invitrogen or from New England Biolabs are
used with the corresponding X10 buffers sold by the supplier. The
reactions are carried out for 2 h at 37° C.

[0116] The plasmid PM1816 was constructed from the plasmid pET28c
(Novagen) which was amplified by PCR so as to eliminate the F1 origin.
This amplification made it possible to create the PacI and AscI sites,
and the Cer fragment was then cloned between these same restriction
sites. The vector thus created was named pM1800 (cf. FIG. 5 for a
schematic representation). Finally, the sequence SEQ ID No. 9 (containing
RBS-Omp A-rEPA) was cloned between the XbaI and EcoRI sites, thus
creating the vector pM1816.

[0117] The ligation of the PCR fragments and of the digested vector was
carried out with the rapid DNA ligation kit from Roche in 5 min at
ambient temperature, with a 6× molar excess of fragments. The new
vector obtained is named pM1816+ccdA.

[0118] In order to remove the kanamycin gene, the plasmid pM1816+ccdA was
digested with the ClaI and AscI enzymes. The sticky ends thus freed were
treated with mung bean exonuclease (New England BioLabs.) used at 10
U/μl, at 30° C. for 1 h. The religation of the vector at the
blunt ends was carried out with the enzyme: T4 DNA ligase New England
BioLabs. (400 U/μL) overnight at 16° C., thus deleting 690 by
out of 816 by of the kanamycin gene.

[0123] The SE1 bacteria are derived from BL21λDE3 cells and have the
ccdB poison gene in their genome. They are sold by the company Eurogentec
and are characterized by the following genotype: F-, CmR, ompT, hsdSB
(restriction-, modification), gal, dcm, DE3(lacI, T7 polymerase under the
control of the PlacUV5 promoter) Ion, ccdB+.

[0124] The BL21λDE3 cells, derived from E. coli B cells, are sold by
Invitrogen and are characterized by the following genotype: F- ompT
hsdSB (rB-mB-) gal dcm(DE3).

[0125] The LB medium is commercially available from Invitrogen. It
comprises, per liter of medium, 10 g of tryptone, 5 g of yeast extract,
10 g of NaCl and qs 1L with H2O buffered at pH 7.5 by the addition
of 5N NaOH.

[0126] The prokaryotic cells were transformed by electroporation according
to the conditions below:

[0127] The E. coli BL21λ DE3 cells are incubated for 30 min in ice
after the addition of 8 μL of product derived from the ligation. The
transformation is carried out by heat shock for 45 seconds at 42°
C.

[0128] The transformed bacteria are taken up in SOC medium (comprising,
per liter of medium, 20 g of tryptone, 5 g of yeast extract, 0.5 g of
NaCl, 10 ml of 250 mM KCl solution, 5 ml of 2M MgCl2 solution, 20 ml
of a 1M glucose solution and qs 1L of H2O at pH 7 with 5N NaOH) and
then incubated for 1 h at 37° C. and plated out on LB agar medium
+50 μg/mI kanamycin, before overnight incubation at 37° C.

[0129] The SE1 bacteria are incubated for 10 min in ice after the addition
of 2 μL of the product derived from the ligation. The transformation
by electroporation is carried out using the Gene pulser® machine
(Biorad) in a cuvette comprising 2 electrodes 1 mm apart, under a current
of 1.7 kV. The transformed bacteria are taken up in SOC medium and then
plated out on antibiotic-free LB agar medium, before overnight incubation
at 37° C.

[0130] An expression assay was carried out under the following conditions:

[0131] The preculture is prepared by inoculating 5 ml of LB medium with a
colony picked from a Petri dish onto which the transformed bacteria were
plated out, and incubating overnight at 37° C. with shaking. The
following day, 50 ml of LB or LB+ kanamycin medium are inoculated with
500 μl of said preculture. This culture is shaken at 37° C.,
until the OD at 600 nm is between 0.4 and 0.8. At this stage, the culture
is divided up into 2 times 25 ml in 2 Erlenmeyer flasks. A solution of
IPTG is added to one Erlenmeyer flask at a final concentration of 0.01 M.
The other Erlenmeyer flask will serve as a noninduced control.

[0132] The two cultures are again shaken for 4 h at 37° C. The OD
is then measured, and a 1 ml sample is taken from the 2 Erlenmeyer
flasks.

[0133] The samples derived from the expression assays in E. coli were
taken up in 2× denaturing blue (100 mM Tris-HCl pH 6.8; 20%
glycerol; 4% SOS; 0.2% bromophenol blue; 200 mM β-mercaptoethanol)
in order to concentrate to an OD of 10 per ml. 20 μl were then loaded
onto a 4-20% Tris-glycine gel (PAGEr® Duramide® Precast Gels,
Cambrex). The Invitrogen marker 10748-010 was used as molecular weight
marker.

[0134] After the loading, the gel is subjected to migration for 2 hours in
migration buffer (250 mM Tris-HCl; 1.92M glycine; 1% SDS) at a voltage of
175V. The gel is then either visualized with Coomassie blue (rinsing for
30 min in distilled water, staining for 30 min in

[0136] After electroblotting, the Western blotting membrane is incubated
for 1 hour in a solution of PBS-3% milk. The membrane is then washed
three times in PBS-0.05% Tween before being incubated for 1 hour at
ambient temperature with the first specific antibody corresponding to an
anti-rEPA rabbit serum. The membrane is then washed three times in
PBS-0.05% Tween and then incubated for 1 hour at ambient temperature with
an HRP-conjugated anti-rabbit IgG goat IgG (Zimed ref 65-6120) diluted to
1/1000 in PBS. After 3 washes in PBS-0.05% Tween, the membrane is
incubated for 15 min with the peroxidase substrate ("peroxidase Opti-4CN
substrate kit", Biorad) and rinsed with distilled water.

[0137] The results obtained can be summarized in the following way:

[0138] The amount of rEPA produced with the pSP1 system in the SE1
bacteria is evaluated at 12% (of total proteins) and that produced with
the conventional PM1816 system with kanamycin selection is 14% of total
proteins, i.e. amounts that are entirely equivalent. A strongly revealed
band at approximately 67 kd corresponding to the rEPA protein is noted,
even more strongly in the "induced" samples.

[0139] There is no visible difference under these conditions between the
conventional PM1816 system and the pSP1 system.

[0140] In order to analyze the stability of the plasmid pSP1 in the SE1
bacteria, after culturing for 4 h post-induction, the bacteria are plated
out on LB agar medium. 88 clones thus obtained were subsequently analyzed
as DNA minipreparations.

[0141] All the clones having grown in liquid medium possess the plasmid.

[0142] It can thus be concluded that the ccdA gene is functional, since
the SE1 bacteria cannot survive without the ccdA antidote gene contained
in the plasmid, and that the stability of the plasmid during the
expression assay is very good.

EXAMPLE 3

Expression of the rEPA Protein in 1-Liter and 30-Liter Fermenters

[0143] The objective of this study is to evaluate the vector according to
the invention for the production of recombinant proteins on the pilot
scale.

Production of an Experimental Seed Lot (Glycerol Stock)

[0144] After transformation of the SE1 strain (Eurogentec) with the
plasmid pSp1 as is described in the previous example, the expression
level is evaluated on a small scale.

[0145] 50 ml of LB medium are inoculated at 1:100 with the SE1 cells
containing in the plasmid pSp1, and incubated overnight at 37° C.

[0146] When the OD (600 nm) reaches the value of 0.8, the IPTG (1 mM) is
added for the induction.

[0147] After incubation for 4 h, 1 ml of bacterial suspension is removed
and centrifuged and the pellet is resuspended in 75 μl of 50 mM Tris
buffer, pH 7.4, 1 mM EDTA and 20% sucrose. The volume is then adjusted to
750 μl with 50 mM Tris-HCl buffer, pH 7.4, 1 mM EDTA. The sample is
loaded onto a polyacrylamide gel. After staining with coomassie blue, the
protein bands are scanned with a GS-710 densitometer from Biorad. Under
these conditions, it is observed that the amount of protein of interest
represents 12% of the total proteins.

[0148] After evaluation of the expression on a small scale, an
experimental seed lot comprising 45 vials was prepared according to the
following procedure: the clone evaluated is plated out altogether on
trypcase-soy agar and incubated at 37° C. for 24 h. The bacterial
layer is then taken up with 2 ml of 2×LB medium so as to inoculate
100 ml of modified 2×LB medium in a 500 ml Erlenmeyer flask in
order to obtain an initial OD 600 nm of between 0.1 and 0.2 unit. The
Erlenmeyer is shaken at 175 rpm at 37° C. until an OD 600 of
between 3 and 5 is obtained. The suspension is cooled and then
centrifuged at 4000 rpm for 30 min at 4° C. The supernatant is
removed and the pellet is taken up with 50 ml of pre-cooled freezing
medium. The suspension is then dispensed into nunc cryotubes at a rate of
1 ml per tube, and then stored at a temperature ≦-70° C. in
the presence of glycerol.

[0150] The freezing medium is composed of 50% of modified 2×LB
medium and 50% of a freezing solution. The freezing solution contains,
per liter of mixture, 10 ml of 1M MgSO4, 200 g of 100% glycerol and
5 g of NaCl, qs with UF water. It is sterilized by 0.22 μm filtration.

Evaluation in a Fermenter

[0151] 500 ml of GluSKYE fermentation medium (contains, per 1 L: 40 g of
yeast extract, 3 ml of 1M MgCl2, 0.53 g of K2SO4, 1 g of
NaCl, 0.84 g of K2HPO4, 44 g of glucose, qs with UF water (30
min/121° C.)) are inoculated with 25 ml of a preculture shaken at
175 rpm for 16 h and maintained at a temperature of 37° C. This
preculture containing the 2×LB medium was inoculated beforehand
with 500 μl of a seed lot vial, stored at -70° C. and
containing the producer bacterium in the form of frozen material.

[0152] The fermentation equipment used is the Biostat Q (Sartorius), which
comprises four 1 L (500 ml working volume) tanks connected to a
regulating cabinet and to a supervision system which records and archives
the data. The fermentation parameters are the following: pH 7.0;
temperature 37° C., initial shaking of 200 rpm, initial aeration
of 0.5 L of air/min, pO2 maintained at 30%. The induction of the
r-EPA protein is carried out by addition of 1 mM IPTG when the OD at 600
nm is between 25 and 35 units. The culture is subsequently stopped after
3 h of induction.

[0153] A variant of the method consists in inducing the rEPA synthesis at
an earlier stage of the culture (between 1 and 3 OD units instead of
25-35), in order to test the stability of the plasmid under more
selective conditions. In the same context, another variant consists in
prolonging the duration of induction to 20 h.

[0154] A first evaluation was carried out in Biostat Q fermenters (BBI)
configured as 4 tanks having a working volume of 0.5 liter, which can be
individually parametered with respect to pH, pO2 and temperature.
The composition of the medium and also the value of the physical
parameters are identical to those selected for the method based on the
use of kanamycin. Furthermore, 4 cultures are carried out; the control
strain E. coli BL21/pM1816 (Km system) and the strain of the new system
E. coil SE1/pSP1 are tested under 2 induction conditions (rapid induction
and late induction).

[0155] The evaluation on a larger scale was carried out using an Applikon
30L fermenter, adhering to the same principles.

[0157] Each culture sample is treated by osmotic shock so as to release
the product present in the periplasm. Each extract is then analyzed by
PAGE.

Monitoring of the Stability of the Plasmid:

[0158] The monitoring is carried out by individual analysis of the plasmid
content of each colony. Starting from culture samples diluted and
deposited on a dish, after incubation, about a hundred colonies are taken
up individually in microplates (96 wells) containing LB medium. The plate
is then placed in an automated plasmid extraction system (Qiagen
bioroBot® Systems) for incubation for 24 h so to allow amplification
of the bacteria. They are subsequently centrifuged, lysed and filtered,
and the samples are then loaded onto an agarose gel for electrophoresis.
The presence of plasmid on the gel is verified after migration and
staining.

[0159] In the case of the PM1816 system, 100 individual colonies are
subcultured on LB agar medium supplemented with kanamycin at a final
concentration of 50 μg/ml.

[0160] The gel of FIG. 12 documents the presence of rEPA undergoing
induction.

[0161] With the new system, the induction can be maintained for 24 h
without significant loss of plasmid, even if the induction with IPTG is
carried out at the beginning of culturing. Under these same rapid
induction conditions, only 5% of the cells retain the plasmid with the
system based on Km selection, as shown in the table below:

[0162] Productivity of rEPA in 1 L fermenter after 24 h of culture
[0163] % P+ measures the percentage of cells containing the plasmid among
the total viable cells. [0164] the amount of rEPA is evaluated by ELISA
as is described in the previous examples.

Evaluation in a 30 l Fermenter:

[0165] The new construct was subsequently tested at the standard clinical
batch production scale, i.e. 30l. The culture parameters remain the same
as those described in the preceding cultures. The volume of culture
medium in the fermenter is 20 liters. The rEPA production kinetics are
given in FIG. 13.

[0166] We evaluated the new vector for the production of a recombinant
protein. This system does away with the presence of an
antibiotic-resistance gene and therefore no longer has recourse to the
use of antibiotics whatever the culture step. The pilot-scale evaluation
in a 30 l fermenter showed that the plasmid is maintained throughout the
culture, without any loss being observed.

[0167] The productivity of protein of interest obtained is greater than
that observed with the conventional system using an antibiotic-resistance
gene as selectable marker.

EXAMPLE 4

Analysis of the Advantage of the Cer Fragment

[0168] In order to evaluate the contribution of the Cer fragment to the
increase in production observed, various vectors were constructed.

[0169] The vector pM1800 was amplified by PCR in order to remove the
kanamycin-resistance gene and to create the PstI and Acc65I restriction
sites.

[0170] The SEQ ID No. 8 cassette was constructed by combined PCRs from the
vector pM1800.

[0171] First of all, the mob promoter and the 5' part of the ccdA gene
were amplified from the plasmid pStaby (Delphi gentic). The PCR primer
provides a fragment of the 3' part of the kanamycin gene. This PCR is
named C1.

[0172] A second PCR makes it possible to amplify the 3' part of the ccdA
gene. The PCR primer provides a fragment of the 5' part of the kanamycin
gene. This amplification is named C2.

[0173] The third PCR concerns the amplification of the kanamycin gene from
pM1800. The 5' primer provides a fragment of the 5' part of the ccdA
gene. The 3' primer provides a fragment of the 3' part of the ccdA gene.
This PCR is named C3.

[0174] PCR C1 is combined with PCR C2 and the product is subsequently
combined with PCR C3. The fragment obtained is digested with the PstI and
Acc65I enzymes and cloned into the vector pM1800, digested with the same
enzymes.

[0175] The ligation of the two fragments was carried out with the enzyme:
T4 DNA ligase, New England BioLabs. (400 U/μL), overnight at
16° C. The new vector created is named pSP301. A representation of
this vector is given in FIG. 6.

[0176] A version of this vector without the cer fragment was constructed.
The pSP301 vector was digested with the PacI and AscI enzymes, thus
deleting the cer fragment. The sticky ends thus freed were treated with
mung bean exonuclease (New England BioLabs) used at 10 U/μl at
30° C. for 1 h. The religation of the vector at the two blunt ends
was carried out with the enzyme: T4 DNA ligase, New England BioLabs (400
U/μL) overnight at 16° C.

[0177] The new vector thus constructed is named pSP2. A schematic
representation of the plasmid pSP2 is given in FIG. 7.

[0178] The SEQ ID No. 9 cassette (containing RBS-Omp A-rEPA, 2024 bp),
isolated from the plasmid pM1816, was cloned into the pSP301 and pSP2
vectors between the XbaI and EcoRI sites. The kanamycin gene was
subsequently excised from these vectors by homologous recombination as
described in Example 4. The plasmids obtained are named respectively pSP6
and pSP4, a schematic representation of which is given in FIGS. 8 and 9.

[0179] The SE1 strain was transformed with the pSP6 and pSP4 vectors and
the BL21ADE3 strain was transformed with the pM1816 vector. A comparative
expression assay was carried out using these 3 vectors. The same working
conditions as those of Example 2 were used.

[0180] The best rEPA production results were obtained with the constructs
containing the cer fragment.

[0181] In order to reinforce the first results obtained, the same type of
study was carried out with a second antigen: the Helicobacter pylori AlpA
protein.

[0182] The following vectors were constructed.

[0183] The AlpA protein gene (SEQ ID No. 4) was cloned into the pSP301 and
pSP2 vectors (previously described) between the NcoI/XabaI sites. The
kanamycin gene of these new plasmids is excised by homologous
recombination, as described in Example 4, thus creating respectively the
pSP5 and pSP3 vectors. A schematic representation of these vectors is
given in FIGS. 10 and 11.

[0184] The SE1 strain was transformed with the pSP5 and pSP3 vectors and
the BL21λDE3 strain was transformed with the pMH.P3.1 vector. A
comparative expression assay was carried out using these 3 vectors. The
same working conditions as those of Example 2 were used.

[0185] The results obtained show that the amount of protein of interest
produced (% expressed relative to the total proteins) is greater with the
"no antibiotic" vectors containing the Cer sequence. The densitometric
analysis of the bands obtained by SDS PAGE electrophoresis clearly
indicates a decrease in expression in the absence of the Cer fragment.

[0186] The comparison of the various constructs shows that a "no
antibiotic" +cer vector results in productivities that are much higher
than those obtained with an "antibiotic" +cer vector, which results in a
greater productivity than that obtained with an "antibiotic" vector
without Cer.

[0187] Furthermore, it is observed that the "no antibiotic" +cer vector
results in an increase in production of the protein of interest of at
least 10% compared with an "antibiotic" system without cer.

EXAMPLE 4

Deletion of the Kanamycin Gene by Homologous Recombination

[0188] The pSP301 or pSP2 vectors are digested with the ClaI enzyme.

[0189] 2 μg of plasmid DNA are digested with 6U of ClaI enzyme, for 1 h
at 37° C. 100 μl of electrocompetent CYS21 bacteria (Delphi
Genetics) are then transformed in 200 ng of this digestion. The entire
transformation is plated out onto an LB plate.

[0190] The clones obtained are subsequently analyzed as DNA
minipreparations by enzymatic digestion.

[0191] The analysis of clones obtained after transfection of
electrocompetent CYS21 bacteria with the linearized DNA shows that all
the colonies obtained contain a recombined plasmid. The sequencing of 2
clones taken randomly made it possible, in addition, to confirm that the
assembly of the ccdA gene was in accordance with the expected result.

[0192] This homologous combination was carried out with the plasmids pSP3,
pSP5 and pSP4, pSP6.

[0193] The homologous recombination method provides a positive selection
of the recombined clones insofar as the ccdA gene can be functional only
after elimination of the kanamycin-resistance gene. We can thus be
certain that the transformed bacteria have lost the kanamycin-resistance
gene, which is not the case when the system based on an enzyme digestion
step followed by religation is used. This method therefore has the effect
of completely doing away with any additional analysis aimed at
documenting the absence of the kanamycin-resistance gene. This additional
analysis will conventionally be carried out on a plasmid DNA preparation
analyzed by restriction mapping or alternatively by means of the specific
PCR amplification method.